A wide variety of industrial products and other samples involved in industrial processes (raw materials, in-process samples, environmental samples etc.) need to be tested for microbial contamination. In some cases the final product must be sterile; in other cases a limit is set on the total number of micro-organisms allowed. Often tests are performed for the presence of certain specific organisms, and again the requirement may be absence in a particular amount of sample or there may be a limit on the number allowed.
Traditionally these tests involve use of culture techniques. However, these traditional testing methods are slow. Typically, it usually takes at least 24 hours before healthy, fast-growing bacteria or yeasts form colonies large enough to comfortably count on an agar plate. However, many samples contain stressed micro-organisms that need a recovery period before they begin to multiply, or organisms (including molds) which grow slowly on common types of culture media. Therefore many validated culture methods require an incubation period of 24-48 hours and, in some cases, 5 days or more.
Many microbiological tests are now performed much more rapidly using an ATP bioluminescence reaction to detect ATP released from a cell (e.g., a microorganism cell). ATP is an essential part of energy metabolism and therefore an indicator of the presence of living organisms or other organic matter.
These tests make use of the luciferase enzyme. A bioluminescence reagent contains luciferase with; inter alia, its substrate luciferin, magnesium ions and a suitable buffer. When adenosine-5′-triphosphate (ATP) is added to this reagent, luciferase catalyzes the emission of light. The result of the test is recorded as an RLU (relative light unit) value. The RLU value generally is proportional to the quantity of microorganisms present in a test sample.
Certain samples (e.g., milk) may contain relatively high levels of free ATP associated with casein micelles and ATP present in somatic cells. In these situations, chelating agents can be used to disrupt the micelles, mild detergents can be used to lyse the somatic cells, and the “nonmicrobial” ATP can be hydrolyzed using apyrase (an ATP-hydrolyzing enzyme); thereby enabling the detection of microbial ATP. There remains a need for simple, convenient tests to detect microbial ATP.